extra unknown band after restriction digestion?

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    • #16680
      genetherapy
      Participant

      I have a vector size 4775 bp and insert 500 bp. I ligated them before and yesterday I digested with related restriction enzymes for my experiment. But interestingly I saw 3 bands. One is insert(500 bp) the other is vector (4775 bp) and there is an other band bigger than 10000bp (My marker’s bigger size is 10000). Today I digested again and saw the same bands. I use stock pellets. I mean, I purify plasmid DNA from bacterial pellet and then I do restriction digest. Could it be the bacterial DNA if I could not purify well?? But E.coli DNA is 4 million bp. Could anyone give some advice.
      Greatly appreciated.

    • #111855
      JackBean
      Participant

      If you’d digested E.coli gDNA, you would probably get more than one band. Are you sure there was no background smear?

    • #111859
      genetherapy
      Participant

      I didn’t digested e.coli DNA. I purified plasmid dna from e.coli and then digested plasmid DNA. Could it be the e.coli DNA if I could not purify plasmid DNA from bacteria (top 10 e.coli) well. my biggest marker is 10 kb and this extra band is bigger than 10 kb.

    • #111860
      genetherapy
      Participant

      I forgot to say: yes, there was not any smear. Only a band bigger than 10 kb.

    • #111862
      JackBean
      Participant

      Did you check uncut plasmid?

    • #111863
      genetherapy
      Participant

      Yes, I checked it. It also have the same extra band

    • #111864
      genetherapy
      Participant

      And yesterday I loaded little amount and looked the gel,did not see any smear but today a loaded whole restriction reaction to gel and checked it and realised have a upward smear. Also have the extra band. Downward smear means degredation but what does upwaard smear. Still thinking about bacterial contamination??? I really need help. Attached the gel view.


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    • #111865
      JackBean
      Participant

      But otherwise it’s distinct from the cut DNA, right?

      The picture is not working. Try different format or upload it directly e.g. to tinypic.com

    • #111866
      genetherapy
      Participant

      <a href="http://tinypic.com?ref=123oztv&quot; target="_blank"><img src="http://i49.tinypic.com/123oztv.jpg&quot; border="0" alt="Image and video hosting by TinyPic"></a>

    • #111867
      genetherapy
      Participant

      attached other format of the view


      Attachments:

    • #111915
      genetherapy
      Participant

      Nobody has an idea??

    • #111946
      canalon
      Participant

      NOt sure what we are looking at in each lane. But my guess, since it appears that you have loaded a ton of DNA in your gel (considering the look of the band), would be trace amount of genomic DNA.

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